In the rearing of livestock, it is very important to accurately determine the pregnancy status of bred animals. In particular, it is the accurate and early identification of failed pregnancy of an animal that has been bred that is economically important. Presently, once an animal is bred, for example a cow, pregnancy status is determined by such methods as palpation, which does not provide an accurate determination of pregnancy status until after 30 days following breeding. Because cattle have an estrous cycle of about 21 days, this means that with presently available methods at least one opportunity for breeding an animal that fails to conceive, the estrus period immediately following the failed breeding, will be missed.
This has important economic consequences for the cattle breeding industry, especially for the dairy industry. Efficient milk production farming requires that cows be successfully bred to become pregnant 80-100 days after calving. Dairy cows, however, have a low fertility rate with artificial insemination, requiring, on average, 2.5 to 3 inseminations per conception. Therefore, a significant need exists for a method by which a dairy farmer may accurately determine that an animal is not pregnant without missing an opportunity to re-breed the animal at the next estrus period following an unsuccessful breeding.
Sasser, U.S. Pat. No. 4,705,748, incorporated herein by reference, discloses a method for determining pregnancy by detecting a protein produced by a conceptus. By this method, cattle were determined to be pregnant as early as day 27 following breeding. Sasser does not disclose the diagnosis of pregnancy prior to the time when a subsequent estrus period will have commenced in non-pregnant cattle and does not disclose an early determination of non-pregnancy.
Maternal recognition of pregnancy in ungulates involves local and systemic gene regulation by the conceptus that results in reduced or altered production of the luteolytic signal, prostaglandin F2α ((PGF2α); Yankey et al., Expression of the antiviral protein Mx in peripheral blood mononuclear cells of pregnant and bred, non-pregnant ewes. Journal of Endocrinology 170, R7-R11 (2001); Bazer et al., Regulation of endometrial responsiveness to estrogen and progesterone by pregnancy recognition signals during the peri-implantation period. In Molecular and Cellular Aspects of Peri-implantation Processes, pp 27-47. Ed S. K. Dey. Springer-Verlag, New York, Inc. (1995)). This is in contrast to pregnancy recognition in primates, which involves a direct luteotrophic effect on the corpus luteum (CL) by conceptus-produced chorionic gonadotropin (Bazer et al. 1995). The signal for maternal recognition in ungulates is the secretion by the conceptus of interferon-tau (IFNτ) during the second and third week of pregnancy (Bazer et al., 1995; Godkin et al., J. Reprod. Fert. 65:141-150(1982)). IFNτ prevents increases in endometrial estrogen and oxytocin receptors, to abrogate oxytocin-induced luteolytic pulses of PGF2α, and maintains CL function (Spencer et al., Endocrinology 136:4932-4944 (1995)).
IFNτ is a member of the Type I IFN family, which also includes IFN α, β, and ω (Samuel, Virology 183:1-11 (1991)), and, more recently, interferon δ (Lefevre, F., et al., Biochimie 80:779-788 (1998). IFNτ signaling through the Type I IFN receptor and Janus Kinase (JAK)-signal transducer and activator of transcription (STAT) signal transduction pathway (Stewart et al., Endocrinology 142:98-107 (2001)) induces a number of genes in the ovine uterus including 2′,5′ oligoadenylate synthetase (Johnson et al., Biol. Reprod. 64:1392-1399 (2001)), β2-microglobulin (Vallet et al., J. Endocrinol. 130:R1-4 (1991)), IFN regulatory factor 1 (Spencer et al., 1998), ubiquitin cross-reactive protein (Johnson et al., Biol. Reprod. 62:622-627(2000)), and Mx protein (Charleston and Stewart, Gene 137:327-331(1993); Ott et al., Biol. Reprod. 59:784-794 (1998)). While the functions of many of these proteins in the antiviral response are well characterized, their roles during early pregnancy are not.
Mx proteins are monomeric GTPases, which, depending on the species of animal and type of virus, are potent inhibitors of viral replication (Samuel, Virology 183:1-11 (1991)). The sequences of Mx proteins from various species, including sheep, cattle, pigs, and horses, are publicly available through GenBank and have been assigned GenBank Accession numbers X66093, U88329, M65087, and U55216, respectively. Although the antiviral effects of Mx are generally directed against negative-stranded RNA viruses (e.g. orthormyxovirus), their expression is induced in all cells that possess Type I IFN receptors and has been used to distinguish between bacterial and viral infection (Haller et al., Rev. Sci. Tech. 17:220-230 (1998)). Recently Mx mRNA and protein were shown to be elevated from epithelium (by day 13) to myometrium (by day 15) within the uterine wall in pregnant ewes and levels remained elevated through day 25 (Ott et al., Biol. Reprod. 59:784-794 (1998)). In addition, Mx mRNA levels were elevated in the corpus luteum in response to injections of roIFNτ into the uterine lumen (Spencer et al., Biol Reprod 61:464-470 (1999)).
These results indicated that IFNτ was either: 1) acting directly on all uterine cell types (i.e., epithelial, stromal and myometrial) and on the CL; or 2) inducing substances (cytokines) that have paracrine/endocrine effects on uterine cells and other organs including the ovaries; or 3) affecting components of the uterine mucosal and circulating immune systems which then affect the various uterine cells and CL.
It is impractical, however, to measure the level of Mx protein in uterine tissue as a test for evaluating pregnancy status. Besides being an invasive and time and labor intensive process, the disruption of uterine tissues necessary to determine the uterine levels of Mx would tend to have a deleterious effect on a pregnancy.
A significant need exists for a reliable, reproducible, and non-invasive method for determining pregnancy or lack of pregnancy in domestic livestock.